1. Field of the Invention
This invention relates to apparatus and methods for use in the disinfection of surfaces and in particular to disinfect wounds using a high concentration aqueous ozone and also to methods and apparatus for producing a high concentration aqueous solution of ozone.
2. Present State of the Art
Wounds can be divided into two basic categories, acute and chronic. Acute wounds are caused when damage occurs to external intact skin tissue. This includes surgical wounds, bites, burns, cuts, abrasions, lacerations and more traumatic crush or gunshot wounds. Chronic wounds are associated with endogenous mechanisms connected to a predisposed condition that eventually damages the dermal tissue. Chronic wounds often result when the supply of oxygen and nutrients (perfusion) to tissues is impaired. Reduced arterial supply, venous drainage or metabolic diseases can cause chronic wounds. Leg ulcers, foot ulcers and pressure sores are all examples of chronic wounds.
Hunt et al (Hunt, T. K. and Hopt, H. W. 1997, Wound healing and infection—what surgeons and anaesthesiologists can do. Surg. Clin. North America. Vol 77, p 587-606) state that acute wounds will heal rapidly if blood perfusion is maximised, thus providing the cells of the immune system the oxygen and nutrients necessary to ward off infection. Oxygen is an integral requirement for cell growth, division and wound healing Grief et al (Grief R., Akca, O., Horn, E., Kurz, A., and Sessler, D. J. 2000. Supplemental perioperative oxygen to reduce the incidence of surgical wound infection. The New England Journal of Medicine. Vol 342, p 161-167). It is also critical for the respiratory burst of Polymorphonuclear leukocytes (PMNs), which produce potent anti-microbial compounds. As well as providing the energy for metabolic reactions and hence infection defence mechanisms, oxygen also plays a major role in determining the oxidation—reduction potential of tissues. Bakker (Bakker, D. J. 1998. Severe trauma and infections. Anaesthesia. Vol 53, p 65-67). Wound microbiology and associated approaches) identifies that a low redox potential favours the growth of anaerobic bacteria. Bowler et al (Bowler, P. G. Duerden, D. I., and Armstrong, D. G. 2001. Wound microbiology and associated approaches to wound management. Clinical Microbiology Reviews. Vol 14, No 2, p 244-269) state that a low redox potential will facilitate the development of synergistic aerobic/anaerobic populations.
Wounds often have a diverse array of microflora. The primary pathogens involved in the infection of chronic and acute wounds are thought to be Staphylococcus aureus, Pseudomonas aeruginosa and beta-hemolytic streptococci. These pathogens are aerobic or facultative. However, anaerobic pathogens are often overlooked in wound infection investigations, because they reside deep within the dermal tissue. Anaerobic micro-organism isolation, identification and collection are time consuming and labour intensive. Bowler et al (referred to above) investigated and conclude that there is correlation between the incidence of anaerobic pathogens and the prevalence of infection. Bascom (Bascom, J. U. 1996. Pilonidal care: anerobes as visible villans. European Journal of Surgery. Vol 162, p 351) reports that anaerobic bacteria are the true causative micro-organisms of wound infection and that improved oxygenation of wounds is required to minimise infection.
The polymicrobial nature of wounds has been widely published, however Staphylococcus aureus is considered to be the most problematic bacterium in traumatic, surgical and burn wound infections Bowler et al (referred to above), Tengrove et al (Tengrove, N. J., Stacey, M. C. McGechie, D. F. and Mata, S. 1996. Qualitative bacteriology and leg ulcer healing. Journal of wound care. Vol 5, p 277-280) report that when four or more bacterial groups are present within a leg ulcer, the likelihood of healing is significantly reduced. This finding promotes the hypothesis that microbial synergy occurs within wounds increasing the net pathogenic effect and severity of the infection. Oxygen consumption by aerobic bacteria induces tissue hypoxia and lowers the redox potential, which provides a more favourable habitat for anaerobic organisms. Nutrients produced by one micro-organism may encourage the growth of potentially pathogenic co-habiting micro-organisms. Some anaerobes are able to impair host immune cell function and hence provide an advantage for themselves and other co-habiting micro-organisms. Bowler (Bowler, P. G. 2002. Microbiology of acute and chronic wounds. Facing the challenge of wound management in the 21st Century. Master Misericordiae University Hospital) states that micro-organisms are able to aid each other within a wound. Micro-organisms (especially in biofilms) use a communication mechanism called Quorum sensing. This is a cell density dependent form of communication, facilitating survival in a new harsh environment. They release signalling molecules informing each other of “survival tips” (i.e. produce a specific morphological change or a specific defensive chemical).
Debridement is an integral part of wound healing. The removal of dead and unhealthy tissue is essential to minimise the habitat available for microbial colonisation and allow new tissue formation. Debridement is achieved through physical removal of tissue using a sharp instrument or the application of saline or sterile water. The management of bite wounds involves high pressure irrigation to reduce microbial load.
Historically ozone has been used to disinfect wounds in its gaseous form or dissolved within oil. Direct ozone gas application, intravenous injection, rectal insufflation or autohemo-ozonotherapy are all known methods of medical ozone application. Reference should be made to the following Patent Publications for details of such treatments: RU-2178699, FR-2784388, U.S. Pat. No. 6,073,627.
Aqueous Ozone Hypotheses
1. Disinfection
    1.1. Ozone is highly reactive and decomposes through the formation of free radicals to form molecular oxygen. Free radicals have an unpaired electron in their outer orbital making them highly unstable and reactive. These free radicals comprise hydroxyl, superoxide or ozonide radicals. Ozone micro-organism attack is primarily on the cellular membrane, with damage subsequently occurring to other cell sites. The proposed mechanism of action is thought, in large part, to relate to the olefinic bonds within the micro-organism cell membrane being attacked by ozone to form an ozonide or other decomposition product. The ozonide reacts with enzymes, sulfhydryl groups and aldehydes, releasing peroxyl compounds. The peroxyl compounds further damage proteins, DNA and other structures. The cell is lysed and the cytoplasm dispersed. In essence, the aqueous ozone would be used to reduce the microbiological organisms within the wound.    1.2. Aqueous ozone will be particularly effective against anaerobic bacteria due to their lack of anti-oxidants and other oxidation defence systems. Aerobic bacteria produce anti-oxidants such as superoxide dismutase to prevent cellular damage caused through respiration using oxygen. Anaerobic bacteria do not use oxygen to respire and hence have not evolved advanced anti-oxidants. The removal of anaerobic bacteria will reduce the likelihood of infection Bowler (referred to above).    1.3. Free radical based oxidation is random and hence it will be extremely difficult for a micro-organism to develop resistance to aqueous ozone. Free radical based disinfection does not involve target site specificity. Free radicals will be effective against all micro-organisms, with the killing rate being dependent on, among other things, the prevalence of anti-oxidants within different microbial species.    1.4. A sufficiently long contact period will remove all micro-organisms from a wound bed, creating a sterile environment.2. Debridement    2.1. Aqueous ozone is not cell specific and will attack the wound tissue as well as the micro-organisms. Unhealthy or dead tissue is less well perfused than healthy tissue and as such does not contain as much anti-oxidant or enzymatic agents (superoxide, dismutase, glutathione, macrophage, etc). The unhealthy tissues will mount a far weaker defence against the free radical attack than the healthy tissues and hence will be more prone to damage/rupture/removal than healthy tissues. Hence, the aqueous ozone will provide a quasi-selective chemical debridement system, creating an improved healing environment.    3. Moist Healing Environment    3.1. The application of aqueous ozone will provide a moist healing environment (in conjunction with 1.4.). A moist healing environment is critical to wound healing Winter (Winter, G. D. 1962. Formation of scab and the rate of epithelization of superficial wounds in the skin. Nature. Vol 193, p 293-294).4. Reactive Oxygen Species (ROS)    4.1. Aqueous ozone produces Reactive Oxygen Species (ROS) as decomposition intermediaries. The ROS produced will complement the bodies own natural defence system in which polymorphonucleocytes (PMNs) produce ROS to remove micro-organisms. The aqueous ozone healing system is biomimetic, providing a “booster” when the bodies own PMNs have been overwhelmed by infection.    4.2. Aqueous ozone will act as an ROS generator in poorly perfused ischemic tissues. The lack of perfusion inhibits the body's own production of ROS through a deficiency in nutrient/oxygen/energy. The aqueous ozone artificially creates the body's natural infection removal mechanism.    4.3. ROS will support the formation of blood vessels (angiogenesis) and stimulate collagen production (Sen, C. K., Khanna, S., Babiar, B. M., Hunt, T. K., Ellison, E. C., and Roy, S. 2002. Redox control of wound repair. JCB (paper in press) Manuscript M203391200).    4.4. Micro-organisms communicate through quorum sensing, which is facilitated through the release of signalling molecules. ROS may actively oxidise these signalling molecules reducing synergistic survival effects. This mechanism would be important in reducing any biofilm formation.5. Oxygenation    5.1. Aqueous ozone decomposes to water and oxygen. The decomposition reaction takes place within the wound providing surface application of oxygen to cells and produces a hyperoxic environment. Anaerobic bacteria cannot survive in a hyperoxic environment, reducing infection.    5.2. A hyperoxic environment produced through aqueous ozone application can provide a source of oxygen to poorly perfused tissues (ischemic), which may improve wound healing.    5.3. Cytokines and growth factors show an improved mechanistic action in a hyperoxic environment, which can be facilitated through the use of aqueous ozone application equipment.    5.4. The aqueous ozone application equipment contains an oxygen concentrator that can be used to provide high pressure sterile oxygen to a wound. Oxygen is critical to the wound healing process. The equipment allows the application of oxygen to the wound via a high pressure jet or through the use of a hyperbaric chamber around the wound area.6. Acute Wound Response    6.1. Research has identified that inflicting an acute wound within a chronic wound can induce a wound healing response. The cellular oxidation caused by aqueous ozone application may induce an acute wound type response within a non-healing chronic wound.Ozonated Water
Ozonated water is widely used to kill bacteria and other micro-organisms. However, when generating and dissolving ozone in water it is usual to expect levels of under 1 ppm.
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US-A-2002139755 discloses a method for enhancing dissolution of gasses in liquids. The method uses a plurality of nozzles sized and sited to produce micro-fine bubbles and initiate rotational flow.
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